Structure of Temperature-Dependent Allele in T4 Model
Background'''M.G. Grutter et. al. 1987, Structural Studies of Mutants of the Lysozyme of Bacteriophage T4, http://www.ncbi.nlm.nih.gov/pubmed/3681997:' This research group sought to understand how specific amino acids contribute to overall protein stability. This area of study had been pursued by many other molecular biochemists and enzymologists at the time, and continues to be studied in increasing complexity by contemporary researchers. At the time of this study, there was relatively little known regarding the specific molecular effects of single amino acid substitutions on protein stability. It was known that protein folding is exothermic, and influenced by a relatively small change in free energy, due primarily to the hydrophobic effect. The implications of a small free energy change for protein folding is that there exists a sensitive balance between the folded and unfolded states. Small changes in temperature, for instance, has the potential to exert substantial control over whether a wild-type protein is in the folded or unfolded state. Furthermore, it was recognized that a single amino acid substitution outside of the active site had the potential to significantly change the stability of a protein, while leaving the enzymatic activity largely unchanged from a wild-type protein. It was postulated that the molecular basis for the effect of an amino acid substitution on protein stability was most likely a result of the mutant residue's interactions with neighboring amino acid residues. This focus on the importance of site-specific interactions contrasts significantly with the idea of one amino acid substitution causing significant changes in total protein structure. Rather, a specific change can affect how stable the total structure is. '''Experimental Rationale'M.G. Grutter et. al. 1987, Structural Studies of Mutants of the Lysozyme of Bacteriophage T4, http://www.ncbi.nlm.nih.gov/pubmed/3681997':' Based on the premise that a site-specific, molecular interaction was the culprit of a temperature-dependent mutant, this research group sought to uncover the specific molecular basis for a mutant's temperature dependence. They used the T4 model bacteriophage to infect Escherichia coli with a gene mutant that codes for a lysozyme that digests the E. coli cell wall during lysis. The gene mutant was a guanine to adenine substitution at nucleotide position 464 resulting in a threonine to isoleucine transformation at amino acid position 157 in the lysozyme. This group aptly named their mutant TI157. Their first key finding is that the single isoleucine amino substitution substantially alters this protein's stability in higher temperatures compared to the wild type that has threonine, without a significant reduction in it's enzymatic properties. Most interesting, using x-ray crystallography, they attributed the cause of the reduced stability to one less water molecular interacting with the folded protein's exterior, specifically with the 157 threonine's gamma hydroxyl group. Their methods included first mutant screens, then traditional DNA and protein sequencing techniques, and finally x-ray crystallographic imaging of purified wild type and mutant lysozymes. The data for these three key steps is described below. Mutant Screen'''M.G. Grutter et. al. 1987, Structural Studies of Mutants of the Lysozyme of Bacteriophage T4, http://www.ncbi.nlm.nih.gov/pubmed/3681997:' This research group chose to study a lysozyme because previous research groups found that lysosomal proteins are thermodynamically stable ''in vitro. Additionally, the enzymatic activity of the lysozyme can be exploited to create highly observable phenotypic traits. The T4 model system was perfect because one lysozyme was required for cell lysis after infection by T4. The wild-type T4 bacteriophage would only lyse the cells they infected if they had a functional lysozyme. Theoretically, a temperature dependent mutant would lyse the cells up until a certain temperature, at which point the lysozyme would unfold and become non-functional. Following random mutagenesis of the T4 genome, the researchers screened for lysozyme activity in a temperature-dependent fashion. It was found that one mutant retained lysosome activity, indicated by cell lysis, but that the lysozyme activity continually decreased as the temperature increased. They theorized that this decrease in lysosome activity with increasing temperature was a result of reducing functional, and properly folded, enzyme in solution. Interestingly, as shown in table 1 above, the melting point of the mutant lysozyme was decreased, such that there was more enzyme activity at lower temperatures in the TI157 mutant than in the wild type. The most significant decrease in the melting temperature in the TI157 mutant lysozyme from the wild type was at pH2. The optimal temperature of the mutant lysozyme activity was found to be around 30 degrees Celsius. In the figure to the right, from left to right, data from a temperature-dependent assay analyzing lysozyme activity are shown. Lysozyme activity is indicated by a large gray circle around the E. coli cells (the block dot). the columns represent 31, 37 and 43 degrees Celsius. The first row (a,b,c) are E. coli following infection with the wild type T4 Phage. The second row (d,e,f) are E. coli following infection with the TI157 mutant T4 phage. The lowest row (g,h,i) are E. coli following infection from a T4 phage carrying a non-temperature dependent (i.e. non-reversible) mutant of arginine to histidine in the lysozyme protein. These data show increasing lysozyme activity with increasing temperature in the wild type (a,b,c), decreasing lysozyme activity with increasing temperature in the TI157 mutant (d,e,f), and no lysozyme activity in the completely dysfunctional mutant(g,h,i). DNA and Protein Sequencing'''M.G. Grutter et. al. 1987, Structural Studies of Mutants of the Lysozyme of Bacteriophage T4, http://www.ncbi.nlm.nih.gov/pubmed/3681997:' Using the chain-termination method, i.e. Sanger-Sequencing, they identified the DNA differences between the TI157 mutant, non-temperature dependent mutant (used as a control) and the wild type. Then, using EcoRI and HindIII digestion, the T4 genome was transferred into a bacterial plasmid vector, and cloned into a new bacteria host. Via this method, a precise location of the nucleotide substitution was determined by using a temperature-based, lysozyme activity dependent assay. Using a Trypic-digest they identified the amino acid sequence of the lysozyme peptide. '''X-Ray Crystallography'M.G. Grutter et. al. 1987, Structural Studies of Mutants of the Lysozyme of Bacteriophage T4, http://www.ncbi.nlm.nih.gov/pubmed/3681997':' The TI157 mutant lysozyme protein was isolated from the cloning host, purified and crystallized for x-ray crystallography analysis. The specific method of crystallography was precession and oscillation photography. The highest resolution reached was between 1.7 and 6 angstrom. They found a decreased electron density of the hydrophilic solvent in the 157 isoleucine "pocket" of the the TI157 mutant, compared to the 157 threonine "pocket" of the wild type lysosome. They attributed this specific finding to the decreased thermodynamic stability of the TI157 mutant. The reduction of water, on the outside of the folded enzyme, reduces the hydrophobic effect, and therefore decreases change in free energy of folding of the TI157 mutant protein. References: